This invention relates to chemically synthesized ribozymes, or enzymatic nucleic acid molecules and derivatives thereof.
The following is a brief description of ribozymes. This summary is not meant to be complete but is provided only for understanding of the invention that follows. This summary is not an admission that all of the work described below is prior art to the claimed invention.
Ribozymes are nucleic acid molecules having an enzymatic activity which is able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence-specific manner. Such enzymatic RNA molecules can be targeted to virtually any RNA transcript, and efficient cleavage achieved in vitro (Zaug et al., 324, Nature 429 1986; Kim et al., 84 Proc. Natl. Acad. Sci. USA 8788, 1987; Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989).
Because of their sequence-specificity, trans-cleaving ribozymes show promise as therapeutic agents for human disease (Usman & McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037). Ribozymes can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the mRNA non-functional and abrogate protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited.
Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Table I summarizes some of the characteristics of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
The enzymatic nature of a ribozyme is advantageous over other technologies, since the effective concentration of ribozyme necessary to effect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base-pairing. Thus, it is thought that the specificity of action of a ribozyme is greater than that of antisense oligonucleotide binding the same RNA site.
Chemically-modified ribozymes can be synthesized which are stable in human serum for up to 260 hours (Beigelman et al., 1995 supra) and maintain near wild type (the chemically unmodified equivalent of a modified ribozyme) activity in vitro. A number of laboratories have reported that the enhanced cellular efficacy of phosphorothioate-substituted antisense molecules. The enhanced efficacy appears to result from either i) increased resistance to 5'-exonuclease digestion (De Clercq et al., 1970 Virology 42, 421-428; Shaw et al., 1991 Nucleic Acids Res. 19, 747-750), ii) intracellular localization to the nucleus (Marti et al., 1992 Antisense Res. Dev. 2, 27-39), or iii) sequence-dependent non-specific effects (Gao et al., 1992 Molec. Pharmac. 41, 223-229; Bock et al., 1992 Nature 355, 564-566; and Azad, et al., 1993 Antimicrob. Agents Chemother. 37, 1945-1954) which are not manifested in nonthioated molecules. Many effects of thioated compounds are probably due to their inherent tendency to associate non-specifically with cellular proteins such as the Sp1 transcription factor (Perez et al., 1994 Proc. Natl Acad Sci. U.S.A. 91, 5957-5961). Chemical modification of enzymatic nucleic acids that provide resistance to cellular 5'-exonuclease and 3'-exonuclease digestion without reducing the catalytic activity or cellular efficacy will be important for in vitro and in vivo applications of ribozymes.
Modification of oligonucleotides with a 5'-amino group offered resistance against 5'-exonuclease digestion in vitro (Letsinger & Mungall, 1970 J. Org. Chem. 35, 3800-3803).
Heidenreich et al., 1993 FASEB J. 7, 90 and Lyngstadaas et al., 1995 EMBO. J. 14, 5224, mention that hammerhead ribozymes with terminal phosphorothioate linkages can increase resistance against cellular exonucleases.
Seliger et al., Canadian Patent Application No. CA 2,106,819 and Prog. Biotechnol. 1994, 9 (EC B6: Proceedings Of The 6th European Congress On Biotechnology, 1993, Pt. 2), 681-4 describe "oligoribonucleotide and ribozyme analogs with terminal 3'-3' and/or 5'-5' internucleotide linkages".